Biomarker research has exploded, primarily due to the use of proteomics approaches focusing on identifying differences in protein structure and abundance between diseased and normal states. Once identified, these biomarker proteins can be utilized for developing diagnostic tools, and because they are functional molecules, they are also more likely to be valid therapeutic targets.
The accessibility and presence of a large number of proteins in blood plasma make it an excellent matrix in which to search for new biomarkers. However, the estimated dynamic range of various protein concentrations in human serum is up to almost 10 orders of magnitude (Corthals et al., 2000), making the rapid identification of individual disease-associated proteins a tremendous analytical challenge. While total serum protein concentration is approximately 70-90 mg/ml, most useful biomarkers, such as cytokines and prostate specific antigen, are present in the nano to picogram/ml range, and disease-specific changes can be expected to be incrementally small, especially in the early stages of disease (Merrell et al., 2004). Compounding these problems, many disease-specific proteins (e.g. cancer biomarker proteins) are degraded inside the cancer cell by proteolytic enzymes, generating peptide fragments that are subsequently released into the blood. Being low molecular weight in nature, these peptide fragments generally have a half-life of only about two to four hours and most of them are cleared from circulation by the kidney (Lowenthal et al., 2005).
In order to overcome challenges presented by low concentration and rapid turnover of potentially useful cancer peptide fragments, the albumin-associated fraction of proteins and peptides has been investigated as a source of useful new disease-specific biomarkers. Albumin, the most abundant plasma protein (40-50 mg/ml), functions as a scaffold for binding small molecules, lipids, peptides and proteins in the extracellular space. It has been found to form complexes with peptide hormones such as insulin and glucagon; bradykinin, serum amyloid A, interferons, the amino terminal peptide of HIV-1, gp41, and the 14-kDa fragment of streptococcal protein G, among others. Interestingly, it was found that a small percentage of the secreted peptide fragments from degraded cancer proteins have high affinity for serum albumin and form new serum albumin complexes which increase their half-life to about 19 days rather than 2 to 4 hours if they are freely circulating in the blood (Lowenthal et al., 2005). Thus, by their association with serum albumin to form complexes, the longevity of these cancer peptide fragments can be increased by more than 100-fold (Dennis et al., 2002). Due to its high affinity for such a diverse range of ligands, the serum albumin population is expected to be highly heterogeneous, most likely comprising hundreds of different albumin complexes.
Even the most widely used technology for protein separation, 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE), introduced by O'Farrell (1975), cannot separate serum albumin complexes, as it is typically conducted under “denaturing” conditions. Additionally, 2-D PAGE has many other shortcomings including requiring large amounts of samples (about 50 to 100 μg of protein per experiment) and producing a rather streaky and mostly diffused profile when serum sample is analyzed. Furthermore, proteins separated by 2-D PAGE are required to be “blotted” or transferred onto blotting membranes such as polyvinylidene difluoride (PVDF) for Western blot analysis. The efficiency of protein blotting is also variable.
As described in WO 2011/008746, the present inventors developed a new electrophoresis procedure that separates serum protein complexes directly on the PVDF membrane, thus bypassing the cumbersome, time-consuming gel electrophoresis and its subsequent blotting steps (Chang and Yonan, 2008; Chang et al., 2009). The separation of albumin complexes in the present inventors' 2-D High Performance Liquid Electrophoresis (2-D HPLE) is based on their net charge or isoelectric points (pI). The association of a newly released cancer peptide fragment with a pre-existing albumin complex changes its pI and this new albumin complex migrates to a different location on the PVDF membrane, allowing its detection among hundreds of already present albumin complexes. Because it focuses on disease specific peptide fragments, the technique enables not only the identification of new cancer protein biomarkers, but also identifies the cancer peptide motifs within these proteins. When LC-MS/MS analysis is preceded by fraction separation using 2-D HPLE, its dynamic range is enhanced to the 1010 range required for detecting low copy number cancer biomarkers, a sensitivity that has not previously been achieved using other protein separation techniques.
In the United States, pancreatic cancer is the fourth leading cause of cancer-related death in both men and women. It is estimated by the National Cancer Institute that in 2015 more than 48,000 people in the United States will be diagnosed with pancreatic cancer and more than 40,000 will die of this disease. Pancreatic cancer incidence and mortality rates are higher in men than in women. African Americans also have higher rates of pancreatic cancer incidence and mortality than whites or other racial/ethnic groups.
Early stage pancreatic cancer is asymptomatic, and there is no routine screening test for pancreatic cancer. Because pancreatic cancer usually is diagnosed at an advanced stage, the survival rate is extremely low compared with those of many other cancer types. At this time, cancer of the pancreas can be cured only when it is found at an early stage (before it has spread) and only if surgery can completely remove the tumor. Standard treatments for pancreatic cancer include surgery, radiation therapy, chemotherapy, and targeted therapy. It is estimated that approximately $2.3 billion is spent in the United States each year on pancreatic cancer treatment. Serum biomarkers for early detection of pancreatic and other cancers are urgently needed and they will save lives.
It is known that many rapidly growing tumors, including pancreatic cancer, readily become hypoxic due to the inability of the local vasculature to supply an adequate amount of oxygen (Vasseur et al., 2012). The decreased level of oxygen leads to the activation of Hypoxia-inducible transcription factor (HIF-1), which in turn induces processes such as angiogenesis (Dewhirst, 2009). Although angiogenesis occurs in nearly all human solid tumors, it does not occur in an efficient manner, leading to spatial and temporal inadequacies in delivery of oxygen. Therefore, some regions of the tumor may exhibit chronic hypoxia, while other regions of the tumor may undergo cycling hypoxia, by switching between hypoxia and re-oxygenation conditions due to irregular flow of oxygen. The re-oxygenation phase following hypoxia inadvertently causes oxidative stress. Thus, both oxidative stress and hypoxia are common features of tumors that they must deal with.
Oxidative stress is considered as one of the most important risk factors for many diseases in humans, including cancer. Often the oxidative damage resulting from oxidants such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide is found in proteins, lipids and nucleic acids. In proteins, thiol groups are most reactive and easily oxidized to affect enzyme activity or cellular function. Mammalian cells have a battery of oxidation defense systems, including small molecules such as glutathione (GSH) and vitamin E that neutralize the oxidants, and enzymes specialized in oxidant detoxification, such as glutathione peroxidase, glutathione S-transferase and superoxide dismutase. Cancer cells utilize several distinct antioxidant systems to defend themselves against the high oxidative and hypoxic stresses. Antioxidants are molecules that counteract excessive ROS production by preventing or reducing the oxidation of ROS targets. For example, high level expression of antioxidant proteins derived from both thioredoxin and glutaredoxin systems are found in many cancer cells in dealing with ROS induced apoptosis (Karlenius and Tonissen, 2010)
It is also known that free radical induced lipid peroxidation causes a loss of cell homeostasis by modifying the structure and functions of cell membrane. The most important characteristic of lipid peroxidation is to cause the formation of DNA-malondialdehyde (DNA-MDA) adducts by interaction with cellular DNA (uczaj and Skrzydlewska, 2003). Malondialdehyde (MDA) is both mutagenic and tumorigenic. Experimental and clinical studies have shown that a major mechanism for cytotoxic activity of the numerous chemotherapeutic agents is through increased formation of the reactive oxygen species. The chemotherapeutic agents such as cyclophosphamide (cytoxan), doxorubicin (adriamycin) now commonly used for treatment of breast cancer, have all been shown to increase lipid peroxidation and generation of ROS (Moradi et al, 2008).
Vaquero et al (2004) pointed out that one reason why pancreatic cancer is so aggressive and unresponsiveness to treatments is its resistance to apoptosis. They proposed that ROS are a prosurvival, antiapoptotic factor of pancreatic cancer cells. Human pancreatic adenocarcinoma cells generated ROS, which was stimulated by growth factors (serum, insulin-like growth factor I, or fibroblast growth factor-2). Pancreatic cancer cells were also characterized by enhanced NADPH oxidase activity which generates ROS.
Currently, only very limited reports on biomarkers for the detection of early stage cancers are available. For example, we have previously reported the use of a 16 amino acid peptide fragment from the 1,395 amino acid G protein coupled receptor-associated sorting protein 1 (Zheng et al, 2012; Chang and Tuszynski, 2013) as an early biomarker for breast cancer. People are regularly told to watch for early symptoms of cancer. However, by the time symptoms occur, many tumors have already grown quite large and may have metastasized. Moreover, many cancers such as pancreatic and prostate cancers have no symptoms. There remains a pressing need for biomarkers of early stage and late stage cancer to enable the detection, diagnosis, and treatment of cancer at its earliest stages of development, as well as its later stages of development.